Cytotoxic Drimane Sesquiterpenoids Isolated from Perenniporia

Article Cite This: J. Nat. Prod. 2018, 81, 1444−1450

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Cytotoxic Drimane Sesquiterpenoids Isolated from Perenniporia maackiae Jaeyoung Kwon,† Hyaemin Lee,‡ Young Hye Seo,‡,§,⊥ Jieun Yun,∥ Jun Lee,§,⊥ Hak Cheol Kwon,† Yuanqiang Guo,∇ Jong Soon Kang,○ Jae-Jin Kim,□ and Dongho Lee*,‡

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†

Natural Constituents Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 25451, Republic of Korea ‡ Department of Biosystems and Biotechnology and □Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea § Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea ⊥ Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea ∥ Department of Pharmaceutical Engineering, Cheongju University, Cheongju 28503, Republic of Korea ∇ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300353, People’s Republic of China ○ Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea S Supporting Information *

ABSTRACT: A chemical investigation of a basidiomycetes fungus, Perenniporia maackiae, led to the discovery of 12 drimane sesquiterpenoids, including seven new constituents (1−7). The elucidation of the structures was performed via interpreting extensive spectroscopic methods, including ECD calculations. Among all isolated compounds, 1, 2, and 6 exhibited cytotoxicity toward six carcinoma cells, including ACHN, HCT-15, MDAMB-231, NCI-H23, NUGC-3, and PC-3 cells, with half-maximal inhibition of cell proliferation values of 1.2−6.0 μM. Perenniporia maackiae (Bondartsev and Ljub.) Parmasto (family Polyporaceae) is a wood-decay basidiomycetes fungus that causes white rot in various kinds of deciduous trees.1 This fungus has a di- or trimitic hyphal structure with soft and thickwalled basidiospores.1 Previous research has revealed the presence of various compounds, including naphthalenones and diverse terpenoids,2−4 isolated from Perenniporia spp. In particular, drimane sesquiterpenoids, such as pereniporins A and B, have been reported previously as major constituents with antimicrobial and cytotoxic properties.5 These results encouraged further work to elucidate additional structurally related and biologically active constituents from this genus. In a search to discover active constituents with potential anticancer activity, seven new drimane sesquiterpenoids, 6-epi-pereniporin A (1), 6-epi-O-methyl-pereniporin A (2), 6-O-methyl-pereniporin A (3), 3β-hydroxy-6-O-acetyl-pereniporin A (4), 13-hydroxypereniporin A (5), 6-dehydroxy-6-oxopereniporin A (6), and pereniporin C (7), along with five known compounds, pereniporin A,5 6-O-acetyl-pereniporin A,6 ugandensolide,7 11-hydroxycinnamosmolide,8 and 6,9,11-trihydroxycinnamolide,9 were obtained from an EtOAc fraction of a P. maackiae medium, and the elucidation of their structures was performed via spectroscopic experiments. The cytotoxic effects of the compounds against six carcinoma cells were evaluated, and of these, 1, 2, and 6 showed © 2018 American Chemical Society and American Society of Pharmacognosy

activities against all cells tested with half-maximal inhibition of cell proliferation (GI50) values of 1.2−6.0 μM. This report describes the identification and characterization, as well as the biological evaluation, of the isolated constituents from P. maackiae.

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RESULTS AND DISCUSSION Compound 1 was isolated to be a colorless and amorphous solid and assigned the molecular formula C15H24O4 (four unsaturations) via HRESIMS data. The IR spectrum exhibited characteristic absorptions at 3370 and 2940 cm−1, which were indicative of hydroxy and olefinic groups. The 1H NMR data (Table 1) displayed three methyl protons (δH 1.16, 1.09, and 0.95), four methylene protons (δH 4.52, 4.12, 1.77, 1.62, 1.45, 1.39, 1.30, and 1.24), and four methine protons (δH 5.59, 5.25, 4.21, and 1.94). The 13C NMR data (Table 1) exhibited 15 resonances for three methyl carbons, four methylene carbons, including one oxygenated, four methine carbons, including one olefinic and two oxygenated, one oxygenated tertiary carbon, and two quaternary carbons. The occurrence of three distinct Received: February 26, 2018 Published: June 7, 2018 1444

DOI: 10.1021/acs.jnatprod.8b00175 J. Nat. Prod. 2018, 81, 1444−1450

a

18.7, CH2

43.8, CH2

41.8, 50.2, 68.5, 125.3, 139.8, 77.7, 33.3, 97.9, 66.9,

22.3, CH3 36.0, CH3 17.2, CH3

2

3

4 5 6 7 8 9 10 11 12

13 14 15 OCH3

1445

12 8 8, 9, 4, 5, 4, 5, 5, 9, 11 14 13 10

9, 7, 7, 3, 3, 1,

s ddd (12.0, 2.0, 2.0) ddd (12.0, 2.0, 2.0) s s s

5.25, 4.52, 4.12, 1.16, 1.09, 0.95,

HMBC

4, 6, 10, 13, 14, 15

m m m m m m

δH a

1.94, d (9.5) 4.21, m 5.59, m

1.77, 1.24, 1.62, 1.45, 1.39, 1.30,

HMBC data optimized for 8 Hz.

C CH CH CH C C C CH CH2

32.9, CH2

δC

1

position

1

23.5, 34.2, 18.1, 54.2,

42.4, 47.3, 78.6, 121.8, 142.8, 78.3, 36.1, 98.7, 67.8,

44.5,

19.5,

33.6,

δC

Table 1. NMR Spectroscopic Data for Compounds 1−3 in CD3OD

CH3 CH3 CH3 CH3


CH2

CH2

CH2

m m m m m m

5.24, 4.54, 4.14, 1.08, 1.08, 0.95, 3.30,

s ddd (12.5, 2.0, 2.0) ddd (12.5, 2.0, 2.0) s s s s

2.14, d (9.5) 4.00, m 5.79, m

1.77, 1.24, 1.61, 1.45, 1.39, 1.30,

δH

2

9, 7, 7, 3, 3, 1, 6

12 8 8, 9, 4, 5, 4, 5, 5, 9, 11 14 13 10

4, 6, 9, 10, 13, 14, 15

2, 15

HMBC

a

25.1, 33.7, 19.3, 57.0,

39.5, 48.1, 76.3, 120.1, 140.9, 78.4, 35.0, 99.3, 68.0,

45.8,

19.5,

33.0,

δC

CH3 CH3 CH3 CH3


CH2

CH2

CH2

m m m m m m

5.30, 4.54, 4.16, 1.26, 1.04, 1.08, 3.35,

s ddd (12.5, 2.0, 2.0) ddd (12.5, 2.0, 2.0) s s s s

1.92, d (5.0) 3.89, m 5.92, m

1.89, 1.23, 1.68, 1.46, 1.33, 1.28,

δH

3

9, 7, 7, 3, 3, 1, 6

12 8 8, 9, 4, 5, 4, 5, 5, 9,

11 14 13 10

4, 6, 9, 10, 13, 14, 15

HMBCa

Journal of Natural Products Article


Journal of Natural Products

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pereniporin A did an opposite pattern at 208 nm (Δε − 14.3), and the former one was consistent with the computed results, suggesting the absolute configuration at chiral centers as being 5S, 6S, 9S, 10S, and 11R (Figure 3). Therefore, compound 1, termed 6-epi-pereniporin A, was determined to be an epimer of pereniporin A. Compound 2 had the molecular formula C16H26O4 via HRESIMS data, and its 1D NMR (Table 1) suggested a superimposable structure to that of 1. However, the distinction was an additional methoxy group as being present at C-6, as supported via the mass spectrometric data as well as an HMBC cross-peak between OCH3-6 and C-6. The relative and absolute configurations of 2 were demonstrated to be the same as those of 1 according to the NOESY data and ECD calculations (Figures S50 and S51, respectively). Consequently, compound 2 was assigned as 6-epi-O-methyl-pereniporin A. The comprehensive interpretation of the 1D NMR data for compound 3 (Table 1) demonstrated that 3 has the same planar structure as 2, and it was further supported by the same molecular formula C16H26O4 via its HRESIMS data. However, the small coupling constant of J5,6 = 5.0 Hz in 3 indicated a gauche-orientation, as further evidenced by the NOESY data (Figure 2). The measured ECD data of 3 displayed a negative CE at 210 nm (Δε = −12.2), which was opposite to the pattern observed for 1 and 2, and it was found to be quite consistent with the calculated results, indicating the absolute configuration of 3 as being 5S, 6R, 9S, 10S, and 11R (Figure 3). Consequently, compound 3 was assigned as 6-O-methylpereniporin A. The 1D NMR data for compound 4 (Table 2) suggested a structural similarity to that of 3 except for changes in two positions as follows. An additional hydroxy group was observed at C-3, as suggested by the downfield shift in the 1D NMR

methyl groups was indicative of a typical drimane sesquiterpenoid derivative. Given this NMR information, the planar structure was revealed by comprehensive interpretation of a series of the 2D NMR data, including 1H−1H COSY and HMBC NMR spectra (Figure 1). Hence, it was found to be the

Figure 1. COSY and HMBC correlations for compounds 1, 3, 6, and 7.

same planar structure as pereniporin A.5 However, the coupling constant of J5,6 = 9.5 Hz in 1 suggested an anti-orientation, whereas the relatively small coupling constant in pereniporin A was consistent with a gauche-orientation. This distinct coupling constant and the relative configurations determined using the NOESY data supported these compounds as being stereoisomers. The NOESY cross-peaks between H3-14 and H-6, H3-14 and H3-15, and H-11 and H3-15 suggested their locations on the same side, whereas that between H-5 and H3-13 indicated locations on the opposite sides. In particular, a significant NOESY cross-peak between H-11 and H3-15 suggested hydroxy substituents at C-9 and C-11 as being α-oriented according to 3D modeling (Figure 2). The absolute configuration was demonstrated through comparing its measured ECD data with the computed one conducted as referenced in previous reports.10,11 The measured ECD data of 1 exhibited a positive Cotton effect (CE) at 213 nm (Δε + 5.7), while that of

Figure 2. NOESY correlations for compounds (A) 1, (B) 3, (C) 6, and (D) 7. 1446



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Figure 3. Measured and computed ECD spectra of compounds (A) 1, (B) 3, (C) 6, and (D) 7.

spectra [δH 3.15 (H-3) and δC 79.7 (C-3)] and an HMBC cross-peak between H3-13 and C-3. Furthermore, a methoxy group at C-6 was replaced with an acetoxy group, as supported by the appearance and downfield shift of several 1D NMR signals [δH 5.62 (H-6) and 2.03 (H3-17) as well as δC 172.1 (C-16) and 21.7 (C-17)]. The NOESY data suggested the relative configuration of C-3 and the remaining positions to be the same as those of 3 (Figure S50). The absolute configuration of 4 was found to be 3S, 5S, 6R, 9S, 10S, and 11R according to the ECD calculations (Figure S51). Therefore, compound 4 was determined to be 3β-hydroxy-6-O-acetyl-pereniporin A. Compound 5 was found to have the molecular formula C15H24O5 via its HRESIMS data. The 1D NMR data for 5 (Table 2) suggested that it comprises a similar skeleton to that of pereniporin A5 with the difference in the replacement of a methyl group with a hydroxymethyl group, as supported via the 1H NMR data [δH 4.32 (H-13a) and 3.41 (H-13b)] and an HMBC cross-peak between H-5 and C-13. Detailed interpretation of the 2D NMR data enabled the structural elucidation of compound 5 as 13-hydroxypereniporin A. The relative configuration was demonstrated via NOESY correlations,

and the absolute configuration was found to be 4S, 5S, 6R, 9S, 10S, and 11R according to the ECD calculations performed (Figures S50 and S51). The 1D NMR spectroscopic data for compound 6 (Table 2) indicated it to be a drimane sesquiterpenoid derivative similar to pereniporin A. However, a secondary hydroxy substituent at C-6 was oxidized in 6, as indicated by the appearance and downfield shift of 13C NMR signals [δC 202.3 (C-6) and 160.6 (C-8)] attributable to a conjugated α,β-unsaturated carbonyl functionality. After detailed analysis of the 2D NMR correlations in conjunction with HRESIMS data, compound 6 was concluded to be 6-dehydroxy-6-oxopereniporin A. The absolute configuration was shown as 5S, 9S, 10S, and 11R, as determined via the ECD calculations (Figure 3). Compound 7 had the molecular formula C16H26O4 as evidenced via its HRESIMS data. Careful analysis of the NMR data for 7 (Table 2) suggested its structure as being a regioisomer of 3, exhibiting a transposition of the olefinic and methoxy groups. The relative configuration of the asymmetric center was demonstrated according to the NOESY correlations (Figure 2), and the ECD calculations suggested the absolute configuration 1447


1448

a

CH3 CH3 C CH3 2.03, s

HMBC data optimized for 8 Hz.

14 15 16 17 OCH3

28.0, 19.2, 172.1, 21.7,

17.4, CH3

13


40.3, 46.5, 69.2, 119.6, 143.5, 78.2, 39.3, 99.2, 67.5,

4 5 6 7 8 9 10 11 12

3

m m m m dd (11.5, 4.0)

δH

16

2, 15

HMBC

a

δC

33.7, CH2 1.98, 1.30, 18.9, CH2 1.56, 1.44, 42.1, CH2 1.69, 1.24, 39.6, C 2.03, d (4.0) 4, 10, 13, 14 49.2, CH 2.03, 5.62, m 65.0, CH 4.43, 5.61, m 5, 9 123.7, CH 5.70, 140.6, C 78.6, C 39.7, C 5.32, s 9, 12 99.3, CH 5.32, 4.54, ddd (12.5, 2.0, 2.0) 8, 11 67.8, CH2 4.55, 4.17, ddd (12.5, 2.0, 2.0) 7, 8, 9, 11 4.17, 1.11, s 3, 4, 5, 14 68.3, CH2 4.32, 3.41, 1.04, s 3, 4, 5, 13 27.5, CH3 1.11, 1.16, s 1, 5, 9, 10 20.4, CH3 1.21,

2

δC

31.0, CH2 2.13, 1.34, 27.6, CH2 1.71, 1.62, 79.7, CH 3.15,

1

position

4 δH

5 15

HMBC

a

δC

32.5, CH2 2.00, 1.34, 18.5, CH2 1.67, 1.50, 44.1, CH2 1.40, 1.26, 33.2, C d (4.0) 4, 6, 9, 13, 15 56.8, CH 2.98, m 202.3, CH m 123.2, CH 5.72, 160.6, C 78.6, C 45.6, C s 9, 12 98.4, CH 5.37, ddd (12.5, 2.0, 2.0) 7, 8 67.1, CH2 4.69, ddd (12.5, 2.0, 2.0) 7, 8, 9, 11 4.30, d (12.0) 4, 14 22.0, CH3 1.16, d (12.0) 3, 4, 5, 14 s 3, 4, 5, 13 34.3, CH3 1.16, s 1, 5, 9, 10 18.5, CH3 1.06,

m m m m m m

Table 2. NMR Spectroscopic Data for Compounds 4−7 in CD3OD

s s

3, 4, 5, 13 1, 5, 9, 10

2, 10

HMBC

a

δC

δH

7 HMBCa

s d (10.5) d (10.0) s

52.8, CH3 3.36, s

12 8 8, 9, 11 4, 5, 14

8

3, 4, 5, 13 1, 5, 9, 10

9, 7, 7, 3,

dd (3.0, 2.0) 4, 6, 9, 10, 15 dd (10.5, 2.0) dd (10.5, 3.0)

m m m m m m

28.0, CH3 0.97, s 19.2, CH3 0.96, s

31.9, CH2 1.93, 1.27, 27.6, CH2 1.52, 1.49, 79.7, CH 1.46, 1.22, 40.3, C s 4, 6, 10, 13, 14 46.5, CH 2.52, 69.2, CH 6.08, t (2.0) 5, 9, 12 119.6, CH 5.79, 143.5, C 78.2, C 39.3, C s 9, 12 98.1, CH 5.03, dd (15.0, 2.0) 7, 8, 11 67.5, CH2 4.16, dd (15.0, 2.0) 7, 8, 9, 11 3.63, s 3, 4, 5, 14 17.4, CH3 0.94,

m m m m m m

δH

6

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Article

filtered, concentrated, and partitioned with H2O (1.0 L) and EtOAc (3 × 1.0 L). The material obtained from the EtOAc layer (1.2 g) was fractionated by silica gel column chromatography via MPLC (n-hexane/CHCl3/acetone, 1:1:0 to 0:1:3, 35.0 mL/min) to afford 15 fractions (F1−F15). F9 (36.6 mg) was separated by means of semipreparative HPLC (250 × 20 mm i.d., 5 μm, CH3CN/H2O, 1:4 to 1:0, 8.0 mL/min, detection UV 210 nm) to obtain 6-O-acetyl-pereniporin A (4.7 mg, tR = 33.6 min), 11-hydroxycinnamosmolide (2.4 mg, tR = 34.8 min), and ugandensolide (3.0 mg, tR = 35.7 min). F10 (217.0 mg) and F11 (125.7 mg) were integrated and purified via the same HPLC system to give 6,9,11-trihydroxycinnamolide (1.6 mg, tR = 29.0 min), 6-dehydroxy-6-oxopereniporin A (6, 7.3 mg, tR = 34.6 min), and 6-epi-O-methyl-pereniporin A (2, 10.0 mg, tR = 37.6 min). The remaining impure fractions were further purified using the same HPLC system to obtain pereniporin A (54.0 mg, tR = 24.2 min), 6-epi-pereniporin A (1, 12.6 mg, tR = 26.8 min), 6-O-methyl-pereniporin A (3, 3.2 mg, tR = 42.0 min), and pereniporin C (7, 2.1 mg, tR = 46.4 min). F13 (68.1 mg) was separated via above HPLC system (CH3CN/H2O, 1:9 to 7:3, 8.0 mL/min) to give 13-hydroxypereniporin A (5, 3.2 mg, tR = 17.7 min) and 3β-hydroxy-6-O-acetyl-pereniporin A (4, 3.6 mg, tR = 24.3 min). 6-epi-Pereniporin A (1). Yellowish oil; [α]20D +52.4 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (3.56) nm; IR νmax (ATR) 3370, 2940, 1673, 1445, 1385, 1142, 1080, 1006 cm−1; ECD (c 0.20 mM, CH3CN) Δε + 5.7 (213); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 1; ESIMS (negative) m/z 267 [M − H]−; ESIMS (positive) m/z 291 [M + Na]+; HRESIMS (negative) m/z 267.1583 [M − H]− (calcd for C15H23O4, 267.1596); HRESIMS (positive) m/z 291.1585 [M + Na]+ (calcd for C15H24O4Na, 291.1572). 6-epi-O-Methyl-pereniporin A (2). Yellowish oil; [α]24D +9.5 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (3.55) nm; IR νmax (ATR) 3369, 2919, 1691, 1461, 1385, 1118, 1078, 1034 cm−1; ECD (c 0.10 mM, CH3CN) Δε + 10.7 (214); 1 H and 13C NMR (500 and 125 MHz, CD3OD), see Table 1; ESIMS (negative) m/z 281 [M − H]−; ESIMS (positive) m/z 305 [M + Na]+; HRESIMS (negative) m/z 281.1760 [M − H]− (calcd for C16H25O4 281.1753); HRESIMS (positive) m/z 305.1725 [M + Na]+ (calcd for C16H26O4Na, 305.1729). 6-O-Methyl-pereniporin A (3). Yellowish oil; [α]21D −188.7 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 203 (3.52) nm; IR νmax (ATR) 3387, 2920, 1679, 1461, 1362, 1140, 1074, 1026 cm−1; ECD (c 0.07 mM, CH3CN) Δε − 12.2 (210); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 1; ESIMS (negative) m/z 281 [M − H]−; ESIMS (positive) m/z 305 [M + Na]+; HRESIMS (negative) m/z 281.1754 [M − H]− (calcd for C16H25O4 281.1753); HRESIMS (positive) m/z 305.1738 [M + Na]+ (calcd for C16H26O4Na, 305.1729). 3β-Hydroxy-6-O-acetyl-pereniporin A (4). Yellowish oil; [α]21D −111.2 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (3.57) nm; IR νmax (ATR) 3377, 2948, 2878, 1708, 1371, 1246, 1137, 1016 cm−1; ECD (c 0.07 mM, CH3CN) Δε − 12.1 (204); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 2; ESIMS (negative) m/z 325 [M − H]−; ESIMS (positive) m/z 349 [M + Na]+; HRESIMS (negative) m/z 325.1652 [M − H]− (calcd for C17H25O6 325.1651); HRESIMS (positive) m/z 349.1631 [M + Na]+ (calcd for C17H26O6Na, 349.1627). 13-Hydroxypereniporin A (5). Yellowish oil; [α]21D −63.3 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (3.48) nm; IR νmax (ATR) 3336, 2920, 1677, 1419, 1205, 1126, 1043,

of 5S, 8S, 9S, 10S, and 11R (Figure 3), supporting structure 7 for pereniporin C. All drimane sesquiterpenoids obtained were assessed for their ability to inhibit the growth of six carcinoma cells, including ACHN (renal), HCT-15 (colon), MDA-MB-231 (breast), NCI-H23 (lung), NUGC-3 (stomach), and PC-3 (prostate) cells with adriamycin (positive control), and the half-maximal inhibition of cell proliferation (GI50) values are summarized in Table 3. Among these isolates, compounds 1, 2, and 6 exhibited Table 3. Cytotoxic Effects of Compounds 1, 2, and 6 against Six Human Cancer Cell Lines GI50a (μM) compound

ACHN

HCT15

MDA-MB231

NCIH23

NUGC-3

PC-3

1 2 6 adriamycinb

2.0 1.2 3.6 0.07

1.8 1.6 5.4 0.05

2.3 1.5 4.0 0.07

2.1 1.4 4.3 0.08

2.4 1.9 2.3 0.07

6.0 2.0 4.6 0.09

a GI50 is defined as the concentration affording 50% maximum inhibition of cell proliferation and is expressed in each case as the mean of triplicate determinations. bUsed as positive control.

cytotoxicity toward all cells tested with GI50 values between 1.2 and 6.0 μM. In particular, it was noteworthy that compounds 1 and 2 having the 6S-form are active, whereas 3−5 and five known compounds having the 6R-form are inactive. Furthermore, coincidentally, 6 with an oxidized substituent at C-6 was also active, and these results obtained suggested that the functionality at C-6 is important for the mediation of the cytotoxicity of these compounds. Previous studies have shown that numerous cytotoxic drimane sesquiterpenoids, including insulicolide and some polygodial derivatives, are potentially useful bioactive molecules,12,13 and the present findings suggest the necessity of further research, including mechanistic studies.

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EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were obtained using a JASCO P-2000 polarimeter. UV, IR, and ECD spectra were measured on an Optizen POP UV−vis spectrophotometer, an Agilent Cary 630 FT-IR spectrometer, and a JASCO J-1100 spectropolarimeter, respectively. NMR data were obtained via a Varian premium shielded 500 MHz NMR system equipped with a 5 mm AutoX DB probe. ESIMS data were acquired on a Thermo LCQ fleet IT mass spectrometer, and HRESIMS data were recorded on a Waters Q-TOF micro mass spectrometer. Column chromatography was carried out via a Biotage Isolera One MPLC instrument with a SNAP KP-SIL cartridge filled with silica gel (45−50 μm). Semipreparative HPLC was conducted on a Waters instrument consisting of 515 binary pumps and a 2996 PDA detector using a YMC-Pack ODS-A C18 column (250 × 20 mm i.d., 5 μm). TLC was performed on Merck aluminum sheets 60 F254 precoated with silica gel. All solvents used were chromatographic grade. Fungal Material. Perenniporia maackiae was obtained in Guri, Gyeonggi-do, Republic of Korea in July 2011, deposited at Korea University Culture Collection (KUC10246), and identified by Prof. Jae-Jin Kim of Korea University. Extraction and Isolation. Perenniporia maackiae was cultured on PD agar medium of 80 plates at 25 °C for 18 d and extracted with MeOH (3 × 1.0 L) for 7 d. The extract was 1449



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1006 cm−1; ECD (c 0.10 mM, CH3CN) Δε − 4.3 (209); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 2; ESIMS (negative) m/z 283 [M − H]−; ESIMS (positive) m/z 307 [M + Na]+; HRESIMS (negative) m/z 283.1557 [M − H]− (calcd for C15H23O5 283.1545); HRESIMS (positive) m/z 307.1519 [M + Na]+ (calcd for C15H24O5Na, 307.1521). 6-Dehydroxy-6-oxopereniporin A (6). Yellowish oil; [α]21D −71.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 229 (3.77) nm; IR νmax (ATR) 3384, 2926, 1672, 1459, 1361, 1147, 1083, 1016 cm−1; ECD (c 0.4 mM, CH3CN) Δε −10.0 (204), −2.0 (224); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 2; ESIMS (negative) m/z 265 [M − H]−; ESIMS (positive) m/z 267 [M + H]+; HRESIMS (negative) m/z 265.1447 [M − H]− (calcd for C15H21O4 265.1440); HRESIMS (positive) m/z 267.2609 [M + H]+ (calcd for C15H23O4, 267.1596). Pereniporin C (7). White amorphous solid; [α]21D −147.4 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 204 (3.57) nm; IR νmax (ATR) 3435, 2950, 1679, 1460, 1365, 1092, 1047 cm−1; ECD (c 0.07 mM, CH3CN) Δε − 16.0 (205); 1H and 13C NMR (500 and 125 MHz, CD3OD), see Table 3; ESIMS (positive) m/z 305 [M + Na]+; HRESIMS (positive) m/z 305.1723 [M + Na]+ (calcd for C16H26O4Na, 305.1729). ECD Calculations. Conformational searches, optimizations, and ECD calculations were performed based on previous reports.10,11 Cytotoxicity Assays. In vitro cytotoxicity assays were performed based on the sulforhodamine B (SRB) experiment, referring to the United States National Cancer Institute protocol.14 Six different carcinoma cells, including ACHN (renal), HCT-15 (colon), MDA-MB-231 (breast), NCI-H23 (lung), NUGC-3 (stomach), and PC-3 (prostate), were cultured in 96-well plates (2 × 103 cells/well) and treated with different concentrations of the isolated compounds and adriamycin for 72 h followed by fixation using 10% trichloroacetic acid overnight at 4 °C, several rinses with distilled water, air-drying, and staining with a 0.4% SRB solution for 1 h. The unbound dye was rinsed using 1% acetic acid, and the stain was extracted using 10 mM Tris to measure the absorbance at 690 nm via a microplate reader. The half-maximal inhibition of cell proliferation values were calculated via the dose−response curve with a correction for cell count at the start of the experiment.

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Article

ACKNOWLEDGMENTS The research was financially supported by the Korea Institute of Science & Technology Program (2E27831 and 2Z05310), the KRIBB Research Initiative Program, Korea University, and the National Research Foundation of Korea (NRF2015R1D1A1A01060321).

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REFERENCES

(1) Jang, Y.; Jang, S.; Lee, J.; Lee, H.; Lee, H.; Lee, Y. M.; Hong, J.H.; Min, M.; Lim, Y. W.; Kim, C.; Kim, J.-J. Mycobiology 2014, 42, 140−146. (2) Ino, C.; Hirotani, M.; Furuya, T. Phytochemistry 1984, 23, 2885− 2888. (3) Feng, Y.; Wang, L.; Niu, S.; Li, L.; Si, Y.; Liu, X.; Che, Y. J. Nat. Prod. 2012, 75, 1339−1345. (4) Guo, H.; Si, J.; Li, Z.-H.; Feng, T.; Dong, Z.-J.; Dai, Y.-C.; Liu, J.K. J. Asian Nat. Prod. Res. 2013, 15, 253−257. (5) Kida, T.; Shibai, H.; Seto, H. J. Antibiot. 1986, 39, 613−615. (6) Garlaschelli, L.; Mellerio, G.; Vidari, G. Tetrahedron 1989, 45, 7379−7386. (7) Kioy, D.; Gray, A. I.; Waterman, P. G. Phytochemistry 1990, 29, 3535−3538. (8) Madikane, V. E.; Bhakta, S.; Russell, A. J.; Campbell, W. E.; Claridge, T. D.; Elisha, B. G.; Davies, S. G.; Smith, P.; Sim, E. Bioorg. Med. Chem. 2007, 15, 3579−3586. (9) Clarkson, C.; Madikane, E. V.; Hansen, S. H.; Smith, P. J.; Jaroszewski, J. W. Planta Med. 2007, 73, 578−584. (10) Kwon, J.; Lee, H.; Yoon, Y. D.; Hwang, B. Y.; Guo, Y.; Kang, J. S.; Kim, J.-J.; Lee, D. J. Nat. Prod. 2016, 79, 1689−1693. (11) Kwon, J.; Lee, H.; Ko, W.; Kim, D.-C.; Kim, K.-W.; Kwon, H. C.; Guo, Y.; Sohn, J. H.; Yim, J. H.; Kim, Y.-C.; Oh, H.; Lee, D. Tetrahedron 2017, 73, 3905−3912. (12) Tozyo, T.; Yasuda, F.; Nakai, H.; Tada, H. J. Chem. Soc., Perkin Trans. 1 1992, 1859−1866. (13) Fang, W.; Lin, X.; Zhou, X.; Wan, J.; Lu, X.; Yang, B.; Ai, W.; Lin, J.; Zhang, T.; Tu, Z.; Liu, Y. MedChemComm 2014, 5, 701−705. (14) Greten, F. R.; Eckmann, L.; Greten, T. F.; Park, J. M.; Li, Z.-W.; Egan, L. J.; Kagnoff, M. F.; Karin, M. Cell 2004, 118, 285−296.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00175. NMR and HRESIMS data of compounds 1−7, 1H NMR chemical shift as well as structures of five known compounds, and measured and computed ECD spectra as well as NOESY information on 2, 4, and 5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: [email protected]. ORCID

Yuanqiang Guo: 0000-0002-5297-0223 Dongho Lee: 0000-0003-4379-814X Notes

The authors declare no competing financial interest. 1450


Cytotoxic Drimane Sesquiterpenoids Isolated from Perenniporia

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